Quantifying the Extent of Calcification of a Coccolithophore Using a Coulter Counter

Although, in principle, the Coulter Counter technique yields an absolute measure of particle volume, in practice, calibration is near-universally employed. For regularly shaped and non-biological samples, the use of latex beads for calibration can provide sufficient accuracy. However, this is not the case with particles encased in biogenically formed calcite. To date, there has been no effective route by which a Coulter Counter can be calibrated to enable the calcification of coccolithophores—single cells encrusted with biogenic calcite—to be quantified. Consequently, herein, we seek to answer the following question: to what extent can a Coulter Counter be used to provide accurate information regarding the calcite content of a single-species coccolithophore population? Through the development of a new calibration methodology, based on the measurement and dynamic tracking of the acid-driven calcite dissolution reaction, a route by which the cellular calcite content can be determined is presented. This new method allows, for the first time, a Coulter Counter to be used to yield an absolute measurement of the amount of calcite per cell.


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Section 1: Literature views about coccolithophores characterisation measured by a Coulter Counter They translated the coccolith volume to the calcite mass and that was in good agreement with the reported values.

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Section 2: Coccolithophore culturing conditions All three species of coccolithophore were grown on Aquil synthetic ocean water 9 based K/2 culture medium, with F/2 vitamin enrichment 10 (See Table S2). All cultures were maintained by regularly sub-culturing into fresh growth medium under sterile conditions, and during the exponential  In a Coulter Counter experiment a potential is applied across the two platinum foils, consequently electrolysis occurs. In the present case protons are generated at the anode (Eq1), the anode is in the coccolithophore sample and this produced acid is able to dissolve the biogenic calcite (Eq2). (1) For example, for a measurement using an 800 μA current, after one minute of measurement up to 5.0 × 10 -7 mol of protons may be produced. The quantity of protons formed will depend on the efficiency of the electrode process, where in the presence of chloride the electro-generation of chlorine will be a competing process. However, if initially we assume 100% efficiency and taking an example Coulter Counter cell volume of 20 mL, then every minute the proton concentration may increase by up to 25 µM. To experimentally investigate the potential for this adventitiously produced acid to react with the biogenic calcite, a solution containing 4% NaCl, 20 mM CaCl 2 and 1 mM NaHCO 3 adjusted to pH 8 was used as the electrolyte in the Coulter Counter. 0.5mL of the E.huxleyi sample was added to 20mL of the electrolyte.

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The size distribution of the coccolithophore sample was measured using the Coulter Counter 15 times in 40mins. For each run, 1mL of the sample was taken for the analysis and the measurement took ~1min to complete. As shown in Figure S1 the measured volume of coccolithophores decreased as the number of experiments progressed.
If either a larger electrolyte volume is used or if the Coulter Counter cell contains 10 mM Tris buffer then this effect of calcite dissolution can be avoided, which is shown in Figure S2.   On the same sample, we subsequently performed a dynamic Coulter Counter measurement but in the absence of acetic acid buffer. (See details in dynamic assessment in the main text -filter the raw pulses and plot windowed averages of the particle size with time) Figure S5 shows the first 20s of the dynamic particle measurements after sample injected to the electrolyte. Over this timeframe only ~10% of the calcite shell has dissolved. Here the dissolution is driven by the undersaturation of the solution with respect to calcite. S10 Figure S5: First 20s of the calcite shell dissolution in 4% NaCl as measured by the Coulter Counter.

per cell
This section provides details on the derivations and calculations made in the main text.

Dissolution Rate, J Dis (mol s -1 )
Calcite dissolution requires first the acetic acid to diffuse to the surface of the coccolithophores and then the calcium carbonate to dissolve due to acid reaction: [ 3 ] where k is the heterogeneous rate constant (m s -1 ) for the first-order interfacial reaction.
The dissolution reaction is under a mixed kinetics regime, where the mass-transport of the acetic acid and the interfacial reaction both contributes to the overall rate. Natural convection and migration are assumed to be negligible so we only consider diffusion for this case. The total flux for each process is: where D is the diffusion coefficient of acetic acid (m 2 s -1 ), C bulk is the bulk concentration of acetic acid, C surface is the surface concentration of acetic acid, r is the particle radius (m) and R f is the roughness factor -a measure of the surface area of the calcite encrusted surface relative to that of an equivalently sized sphere. In equation 6 we have assumed that the interfacial reacton kinetics for the dissolution follow first order kinetics, this is consistent with the experimental data provide in Figure 4 of the main text and further mirrors work previous reported in the literature. 11 Further, in both equations 5 and 6 we only consider the reaction occur due to direct reaction with the acetic acid. In this work this is reasonable. Experimentally we use a solution that contains 1 mM acetic acid and 10 mM acetate, the acetate serves an important role in increasing the pH of the solution of pH 5.4 and hence minimising the bulk solution phase concentration of free protons. Although protons diffuse almost an order of magnitude faster than acetic acid (D H+ = ~5x10 -9 m 2 s -1 under aqueous high salt conditions) the proton concentration is over two orders of magnitude less than that of acetic acid.
Using steady-state approximation, if we set (7)

Calcite Mass per cell
If the radius r varies linearly with time then, where 100.09 g mol -1 is the molecular weight of calcite, 3.21x10 -6 m and 2.46x10 -6 m is the optical radius with and without calcite shells, 2.50x10 -4 m s -1 is the heterogeneous rate constant for non-absorbing carboxylic acid, 1.01x10 -9 m 2 s -1 is the diffusion coefficient of acetic acid, 1.0 mol m -3 is the bulk concentration of acetic acid. S13 Section 7: Growth curves of three different plankton species Figure S6: Growth curves of three plankton species.
For every 1-2 days, we tracked the cell count of three species via the Coulter Counter from the day of culturing to the day they reached the stationary phase. (Figure S6) For each day, 1mL of the culture is added to 9mL of the diluent (1 in 10 dilution). The diluted culture was then measured by the Coulter Counter three times. 0.1mL of the diluted culture was extracted by the Coulter Counter each time. Cell count shown on the Figure S6 represents the count for the undiluted sample, which equals to the raw cell count measured multiplying 100.
For the acetic acid dissolution experiments, the samples in exponential phase and stationary phase were studied. The E.huxleyi sample reaches the stationary phase on day 7, whereas the G.oceanica and C.braarudii need approximately 12 and 15 days of growth respectively before they stop growing.

Section 8: Expected range of calcite masses reported in the literature
The expected masses of calcites shells for three species is calculated by the product of the number of coccoliths and the average mass per coccoliths. The results are shown in Table S3.